Protein turnover through the ubiquitin-proteasome pathway (UPP) constitutes a major system used by cells to control signaling networks. In this process, proteins are marked with a chain of ubiquitin molecules, which are linked to the target and to each other through isopeptide linkages with lysine residues (Pickart et al. (2004) Biochim. Biophys. Acta. 1695:55; Pickart (2004) Cell 116:181). Ubiquitin is a 76 amino acid protein which becomes linked to lysine residues through its C-terminal glycine residue. Addition of four or more ubiquitin molecules is generally thought to be sufficient for recognition by the proteasome, where poly-ubiquitinated proteins are degraded (Lam et al. (2002) Nature 416:763). Protein turnover through the UPP is known to regulate many diverse cellular functions and to be intimately linked to human disease (Petroski and Deshaies (2005) Nat. Rev. Mol. Cell. Biol. 6:9). Several components of the ubiquitin system have been found to be mutated in cancer. Most frequently, alterations in the UPP system can lead to inappropriate degradation of tumor suppressor and over-expression of oncogenes, thereby promoting uncontrolled proliferation (Cardozo and Pagano (2004) Nat. Rev. Mol. Cell. Biol. 5:739). Defects in the UPP can also contribute to neurodegenerative diseases, such as Parkinson's Disease and the like. While ubiquitination plays a critical role in targeted protein degradation, it also plays important roles in controlling protein localization, the function of membrane proteins, endocytosis and other processes.
Protein ubiquitination is known to involve three major protein activities (Hershko et al. (1983) J. Biol. Chem. 258:8206; Ganoth et al. (1988) J. Biol. Chem. 263:12412). First, ubiquitin is activated to form a high-energy thiol ester bond with an E1 ubiquitin activating enzyme. This process initially involves formation of an ubiquitin adenylate with the C-terminal glycine of ubiquitin (G76), consuming one molecule of ATP, followed by transfer of the G76 carboxylate to the active site cysteine to form a thiol ester (Haas et al. (1982) J. Biol. Chem. 257:10329); Haas et al. (1982) J. Biol. Chem. 257:2543). In the second step, the E1˜Ub thiol ester complex binds an E2 ubiquitin conjugating enzyme through the E1s C-terminal E2-binding domain. Ubiquitin is then transferred from the active site cysteine in E1 to the active site cysteine in the E2 (Pickart et al. (1985) J. Biol. Chem. 260:1573. The E2˜Ub complex then dissociates from the E1 where it can interact with E3 ubiquitin ligases to promote transfer of ubiquitin to lysine residues either in the substrate or on growing poly-ubiquitin chains (Eletr et al. (2005) Nat. Struct. Mol. Biol. 12:933). Thus, the E1 enzyme plays a critical role in the ubiquitination process by allowing ubiquitin activation and facilitating ubiquitin transfer.
There is widespread interest in the use of UPP components as drug targets for disease. Indeed, the first drug targeting the UPP, VELCADE® (Millennium Pharmaceuticals, Inc., Cambridge, Mass.), was approved by the FDA in 2003 (Popat et al. (2006) Expert Opin. Pharmacother. 7:1337). VELCADE® targets the 26S proteasome, thereby blocking degradation of all proteins whose turnover requires the proteasome. VELCADE® has proven useful for the treatment of multiple myeloma. Id. Its mode of action appears to rely on the enhanced sensitivity of certain types of cancer cells for proteasome activity, possibly reflecting an increased requirement for the NFκB pathway, which relies on the proteasome. However, proteasome inhibition is also potentially toxic to normal cells and therefore, obtaining new drug targets with increased specificity would be useful.